Stereoselective Ethynylation and Propargylation of Chiral Cyclic Nitrones: Application to the Synthesis of Glycomimetics

Abstract Ethynylation and propargylation of chiral nonracemic polyhydroxylated cyclic nitrones with Grignard reagents are efficient methods for preparing building blocks containing an alkyne moiety to be used in copper-catalyzed azide alkyne cycloaddition click chemistry. Whereas ethynylation takes place with excellent diastereoselectivity, propargylation afforded mixtures of diastereomers in some cases. The use of (trimethylsilyl)propargyl bromide as precursor of the Grignard reagent is necessary to avoid the formation of undesired allene derivatives. DFT calculations explain, within the experimental error, the observed behavior. Cycloaddition of the obtained pyrrolidinyl alkynes with sugar azides derived from β-(1,3)-glucans provides glycomimetics suitable to be used against fungal transglycosylases.

Scleroglucan is a polymer that forms the fungal cell wall, which consists of a linear β-(1,3)-D-glucose backbone with one β-(1,6)-D-glucose side chain every three main residues cross-linked with chitin through a unit of N-acetylglucosamine. 1 Inhibition of its synthesis causes fungal death and thus enzymes involved in the synthesis and metabolism of scleroglucan are potential targets for developing new antifungal drugs. 2 The development of suitable enzymatic inhibitors of carbohydrate-active enzymes requires depth knowledge of the mode of action of the target enzyme. 3 In this context, tailor-made glycomimetics are of great utility for understanding carbohydrate-protein interactions. 4 Also, it is well known that polyhydroxylated pyrrolidines (iminosugars) are excellent surrogates of carbohydrates mimicking the transition state of several enzymes, mainly glycosyl hydrolases. 5 In a research project focused on the design of polyhydroxylated pyrrolidinyl-derived glycomimetics 1 incorporating β-(1,3)-D-glucose units targeting fungal transglycosylases, 6 we envisaged that the triazole ring, easily accessible through well-known click chemistry 7 and extensively used in glycobiology, 8 would be a suitable linker between the nitrogenated heterocycle and the carbohydrate unit. According to the retrosynthetic approach shown in Scheme 1 where the corresponding carbohydrate azide 2 is easily accessible, it is necessary to develop an efficient methodology for introducing the required triple bond onto the pyrrolidine ring providing key intermediates 3. A well-established synthetic route to 2-substituted polyhydroxylated pyrrolidines consists of a nucleophilic addition to the corresponding cyclic nitrone 4.

Scheme 1 Retrosynthetic analysis for glycomimetics 1
We and others have reported a variety of nucleophilic additions to 4, 9 including allylation, 10 vinylation, 11 alkylation, 12 and hydrocyanation 10c,13 reactions, and in all cases excellent yields and stereoselectivities were obtained. On the other hand, few particular cases have been reported on the direct ethynylation of cyclic nitrones 14 in comparison with the same reaction on acyclic nitrones. 15 Similarly, propargylation of nitrones has also been scarcely explored and only one example has been described. 14d In this paper, we report a novel and stereoselective synthesis of alkynyl and propargyl pyrrolidines 3 from nitrones 4 and demonstrate their utility in the construction of glycomimetics 1.
The starting nitrones 5-7 used in this study were prepared from D-arabinose for nitrones 5 16 and 7, 17 and from Lmalic acid for nitrone 6. 18 The ethynylation reaction was carried out following our previously reported protocol for acyclic nitrones 15 using lithium (trimethylsilyl)acetylide generated in situ at -80 °C (Scheme 2). In all cases, the reaction proceeded smoothly in 20 minutes in good yield and excellent diastereoselectivity, only one diastereomer being detected by NMR spectroscopy. The configuration of the newly created stereogenic center at C-2 was determined to be trans with respect to C-3 of the pyrrolidine ring by NMR spectroscopy (NOESY experiments). The observed diastereoselectivity is in agreement with previous results 10-13 as expected on the basis of steric and stereoelectronic effects. 19 Scheme 2 Trimethylsilyl ethynylation of cyclic nitrones. Reagents and conditions: (i) trimethylsilylacetylene (2.5 equiv), BuLi (2.5 equiv), THF, -80 °C, 20 min.
Propargylation of nitrones 5-7 was trickier than ethynylation. A first attempt consisting of the reaction between nitrone 6 and allenylmagnesium bromide formed in situ from propargyl bromide and magnesium in the presence of mercury(II) chloride, 20 afforded, in a complete diastereoselective way and excellent yield, a 1:2 mixture of the expected product 11 and the corresponding undesired allene 12 (Scheme 3). In fact, the formation of the allenyl derivative had been extensively reported in the propargylation of imines. 21 Hydroxylamines 11 and 12 showed to be very unstable, readily oxidized to nitrones, and were not fully characterized. It is worthy to note that alkyloxy-substituted hydroxylamine derivatives have been obtained by addition of lithiated 1-alkoxyallenes to these nitrones 22 and were highly unstable, easily cyclizing to the corresponding 1,2-oxazine derivatives. On the other hand, it has been reported that propargylation with the Grignard reagent derived from (trimethylsilyl)propargyl bromide afforded the propargyl derivative almost exclusively in most cases. 23 We carried out the reaction of nitrones 5-7 with (trimethylsilyl)propargylmagnesium bromide, 24 formed in situ from (trimethylsilyl)propargyl bromide and magnesium in the presence of mercury(II) chloride, 25 and the corresponding propargyl derivative was obtained as the only product of the reaction (Scheme 4). Surprisingly, in the case of nitrones 5 and 6, a lack of selectivity was observed and mixtures of inseparable hydroxylamines 13 and 14 were obtained, respectively. Thus, nitrone 5 afforded a 3:1 diastereomeric mixture, while with 6 the lack of selectivity was complete (1:1 mixture). Attempts by carrying out the reaction in the presence of Lewis acids that had shown to be useful for modulating stereoselectivity in nucleophilic additions to nitrones, 11a,26 were unsuccessful. Only nitrone 7 afforded enantiomerically pure 15 as the only product of the reaction (Scheme 4). The configuration of the newly created stereogenic center at C-2 was de- S. García-Viñuales et al.

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termined to be trans with respect to C-3 of the pyrrolidine ring in compound 15 by NMR spectroscopy (NOESY experiments). In the case of hydroxylamines 13 and 14, complete identification of the compounds was achieved by selective TOCSY experiments.
In order to rationalize the experimental results observed in both ethynylation and propargylation, DFT calculations at M06-2X/6-311+G(d,p)//M06-2X/6-31G(d,p) level of theory considering solvent effects (PCM model; THF or Et 2 O depending on the reaction studied) were carried out. 27 The ethynylation reaction can be explained by a typical sterical model, similar to that observed in the vinylation of cyclic nitrones, 11 in which the nucleophile attacks preferentially at the less-hindered face. Figure 1 illustrates the transition states corresponding to nitrone 6 for which the difference of 3.0 kcal/mol in favor of Re-TSA1 predicted a trans:cis ratio in good agreement with the experimental observations. The mechanism of propargylation of cyclic nitrones should consider the metallotropic equilibrium between allenyl-and propargylmagnesium bromide, 28 also observed in other metal derivatives such as lithium, 29 zinc, and titanium. 30 The allenyl form is the most stable 31 and there is a prominent tendency to the location of the metal atom at the allenyl carbon, a situation still more favored by the presence of a organosilyl substituent. 32 However, according to the Curtin-Hammett principle, 33 it cannot be discarded as a higher reactivity of the propargyl form of the Grignard reagent. In fact, it is necessary to consider both propargyl and allenyl forms as attacks at the alpha and gamma carbons of the propargyl/allenyl system thus making a total of eight possible transition structures (see Supporting Information). In the case of nitrone 6 and the addition of the unsubstituted Grignard reagent, the two preferred transition states Re-TS-B1 and Re-TS-C1, both corresponding to the attack at the less hindered face (Figure 1), are competitive. When considering all the possible transition structures and the corresponding Boltzmann distribution derived from energy values (see Supporting Information), a 1:2 propargyl:allenyl ratio and a complete trans-diastereoselectivity for both propargyl 11 and allenyl 12 derivatives were predicted in excellent agreement with experimental results. On the other hand, for the addition of trimethylsilyl-substituted Grignard reagent although the propargyl:allenyl ratio is correctly predicted to be 32:1, the lack of diastereoselectivity observed experimentally is not anticipated.
The copper-catalyzed alkyne-azide cycloaddition (CuAAC) in which terminal alkynes are employed does not require protection of the hydroxyl and amino groups, so unprotected alkynyl pyrrolidines were prepared through reduction of the hydroxylamine moiety with Zn/HCl and further desilylation of the triple bond (Scheme 5). Under these conditions compound 17 was obtained from 8. Debenzylation through hydrogenation is not compatible in the presence of the triple bond. Because of it, in the case of

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compound 17, deprotection of hydroxyl groups should be made after the cycloaddition reaction with the corresponding azide. For hydroxylamine 9 a one-pot procedure including further deprotection of the hydroxyl group afforded hydrochloride 19. The 1:1 mixture of hydroxylamines 13 was also treated with Zn/HCl affording a mixture of amines 21a and 21b, which could be separated by flash chromatography. Further desilylation of 21 furnished compounds 22 in good chemical yields. In the case of hydroxylamines 10 and 15, treatment with Zn/HCl afforded complete deprotection of the acetonide moiety leading to compounds 20 and 23.
In general terms, the synthesis of alkynyl pyrrolidines 17, 19, 20, 22, and 23 is experimentally simple, with several one-pot processes and it takes place in good chemical yields thus rendering that sort of building blocks for click chemistry very accessible. Next, glycosyl azides 27-30 derived from D-glucose, D-galactose, and β-(1,3)-D-gluco di-and trisaccharides were prepared from the corresponding peracetylated sugars 24 34 by formation of glycosyl bromides 25, reaction with sodium azide in DMF, and further deacetylation (Scheme 6). The CuAAC reaction was carried out under standard conditions 8a by mixing aqueous solutions of azides and alkynes, a solution of copper(II) acetate, and an excess of powdered metallic copper. After stirring at ambient temperature for 24 hours and purification by ionexchange chromatography, target glycomimetics 31-39 were obtained in good yields (Scheme 7).
In summary, a process for preparing pyrrolidinyl building blocks ready for CuAAC click chemistry is reported. Installation of a triple bond onto a pyrrolidine ring can be done by means of ethynylation and propargylation reactions. Ethynylation reaction proceeded with excellent results in both chemical yield and diastereoselectivity. For propargylation reaction it was necessary to prepare the Grignard derivative from (trimethylsilyl)propargyl bromide; otherwise the allene derivative is the predominant product of the reaction. The different behavior observed in the nucleophilic additions to cyclic nitrones is explained by DFT calculations within the margin of experimental error. An excellent agreement with experimental results was found for the prediction of alkyne:allene ratios. On the other hand, although the high diastereomeric ratios observed in ethynylation and direct propargylation were correctly predicted, the lack of diastereoselectivity observed in the reaction with the trimethylsilyl derivative was not ascertained. The synthesized alkynyl pyrrolidines have been demonstrated to be excellent substrates for incorporating saturated nitrogen heterocycles to glycomimetics. In fact, cycloaddition with glycosyl azides proceeds without the requirement of protecting groups in high yields with complete regioselectivity furnishing the corresponding glycoconjugates. Application of this protocol to other glycomimetics and studies to fully delineate the stereoselectivity of propargylation reactions are currently under investigation in our laboratory.
The reaction flasks and other glass equipments were heated in an oven at 130 °C overnight and assembled in a stream of argon. All reactions were monitored by TLC on silica gel 60 F254; the position of the spots was detected with 254 nm UV light or by spraying with 5% ethanolic phosphomolybdic acid. Column chromatography was carried out in a Büchi 800 MPLC system using silica gel 60 microns. Melting points are uncorrected. 1 H and 13 C NMR spectra were recorded on Bruker Avance 400 instrument in the stated solvent. Chemical shifts are reported in ppm (δ) relative to TMS as external reference. Optical rotations were taken on a JAS-CO P-1020 DIP-370 polarimeter. Elemental analyses were performed on a PerkinElmer 240B microanalyzer or with a PerkinElmer 2400 instrument.
Assignment of NMR signals in compounds 25-39 have been made following the numbering illustrated in Figure 2.

Trimethylsilylethynylation of Cyclic Nitrones; General Procedure A
To a solution of trimethylsilylacetylene (250 mg, 2.5 mmol) in anhyd THF (7 mL) at -10 °C was added n-BuLi (1.6 mL, 1.6 M in hexanes, 2.5 mmol). This solution was stirred for 15 min and then cooled to -80°C . A cold (-80 °C) solution of the corresponding nitrone (1.0 mmol) in THF (20 mL) was then quickly added with a cannula over a period of 15 min. Stirring at -80 °C was continued for an additional 15 min until all the nitrone was consumed (TLC). The reaction was quenched with sat. aq NH 4 Cl (3 mL) and the resulting mixture was allowed to warm to r.t. The reaction mixture was partitioned between EtOAc (15 mL) and sat. aq NH 4 Cl (25 mL), and then shaken vigorously. The layers were separated, and the aqueous layer was further extracted with

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EtOAc (2 × 15 mL). The organic extracts were combined, washed with brine, dried (MgSO 4 ), and filtered. The solvent was removed under reduced pressure and the crude product was chromatographed on silica gel (9:1 hexane/EtOAc) to give the corresponding hydroxylamine.

Propargylation of Cyclic Nitrones; General Procedure B
To a solution of the corresponding nitrone (0.5 mmol) in anhyd Et 2 O (15 mL) at 0 °C was added freshly prepared allenylmagnesium bromide 15 (0.9 mL, 1.75 M in THF, 1.5 mmol). This solution was stirred for 20 min until all the nitrone was consumed (TLC). The reaction was quenched with sat. aq NH 4 Cl (8 mL) and the resulting mixture was allowed to warm to r.t. The reaction mixture was partitioned between EtOAc (20 mL) and sat. aq NH 4 Cl (40 mL), and then shaken vigorously. The layers were separated, and the aqueous layer was further extracted with EtOAc (2 × 20 mL). The organic extracts were combined, washed with brine, dried (MgSO 4 ), and filtered. The solvent was removed under reduced pressure to give a mixture of hydroxylamines.

(Trimethylsilyl)propargylation of Cyclic Nitrones; General Procedure C
To a solution of the corresponding nitrone (1 mmol) in anhyd THF (25 mL) at 0 °C was added freshly prepared (trimethylsilyl)propargylmagnesium bromide 22 (5.5 mL, 0.54 M in THF, 3.0 mmol). The obtained solution was stirred for 20 min until all the nitrone was consumed (TLC). The reaction was quenched with sat. aq NH 4 Cl (10 mL) and the resulting mixture was allowed to warm to r.t. The reaction mixture was partitioned between EtOAc (25 mL) and sat. aq NH 4 Cl (50 mL), and then shaken vigorously. The layers were separated, and the aqueous layer was further extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with brine, dried (MgSO 4 ), and filtered. The solvent was removed under reduced pressure and the crude product was chromatographed on silica gel (85:15 hexane/EtOAc) to give the corresponding hydroxylamine.

Reduction with Zn in Hydrochloric Acid; General Procedure D
To a solution of the corresponding hydroxylamine (0.3 mmol) in 1 N HCl/1,4-dioxane (1:1, 10 mL) was added Zn dust (98.1 mg, 1.5 mmol) and the resulting mixture was stirred for 16 h. Then, the mixture was treated with solid Na 2 CO 3 and 1 M aq NaOH until pH 10 and extracted with CH 2 Cl 2 (2 × 25 mL). The combined organic layers were washed with sat. aq NaHCO 3 , dried (MgSO 4 ), and filtered. The solvent was removed under reduced pressure to yield the crude pyrrolidine.

Desilylation of Trimethylsilylalkynes; General Procedure E
A solution of trimethylsilylalkynyl pyrrolidine (0.24 mmol) in THF (5 mL) at r.t. was treated with Bu 4 NF in anhyd THF (0.24 mL, 1.0 M, 0.24 mmol). After 7 h, the reaction was quenched by the addition of sat. aq NaHCO 3 and the resulting mixture partitioned between CH 2 Cl 2 (15 mL) and H 2 O (25 mL). The layers were separated, and the aqueous solution was extracted with CH 2 Cl 2 (3 × 15 mL). The organic extracts were combined, dried (MgSO 4 ), and filtered. The solvent was removed under reduced pressure and the crude product was chromatographed on silica gel (7:3 hexane/EtOAc) to give the corresponding alkynylpyrrolidine.

Glycosyl Bromides 25; General Procedure F
A solution of the corresponding peracetylated sugar derivative 24a-d (1 mmol) in 33% HBr in AcOH (3 mL) was stirred at r.t. in an ultrasonic bath sonicator for 15 min at which time the reaction mixture was poured into ice-water (50 mL) and neutralized with 3 N aq NaOH. The resulting white solid was extracted with dichloromethane (3 × 30 mL). The combined organic extracts were washed with sat aq NaHCO 3 , dried (MgSO 4 ) and the solvent was eliminated under reduced pressure to give the crude product 25, which was used in preparation of glycosylazides.

Azidation of Glycosyl Bromides; General Procedure G
To a solution of the corresponding glycosyl bromide 25a-d (0.8 mmol) in anhyd DMF (20 mL) was added NaN 3 (156.0 mg, 2.4 mmol) and the mixture was stirred at r.t. for 18 h. Evaporation to dryness afforded a residue that was solved in CH 2 Cl 2 . The solution was washed with water, brine, dried (MgSO 4 ), and the solvent removed under reduced pressure. The crude product was purified by column chromatography.

Deacetylation of Glycosyl Azides; General Procedure H
A solution of the corresponding glycosyl azide 26a-d (0.3 mmol) in MeOH (15 mL) was treated with NaOMe (81.0 mg, 1.5 mmol), and stirred at r.t. for 20 min. After the addition of H 2 O (30 mL), the mixture was desalted with Amberlist 15H + and solvents were removed by evaporation at reduced pressure and finally lyophilized to afford the deacetylated azide as a white foam.

Acknowledgment
This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) (project number CTQ2013-44367-C2-1-P), by the Fondos Europeos para el Desarrollo Regional (FEDER), and the Gobierno de Aragón (Zaragoza, Spain, Bioorganic Chemistry Group, E-10). The authors acknowledge the Institute of Biocomputation and Physics of Complex Systems (BIFI) at the University of Zaragoza for computer time.

Supporting Information
Supporting information for this article is available online at http://dx.doi.org/10.1055/s-0035-1562500. S u p p o r t i n g I n f o r m a t i o n S u p p o r t i n g I n f o r m a t i o n